Challenging protein-nanoparticle interactions. Results with gold nanoparticle and β2-microglobulin system.

The work presented here deals with the investigation of the interaction of gold nanoparticles (AuNPs) with a paradigmatic amyloidogenic protein, namely β2-microglobulin (β2m). The interaction between proteins and nanoparticles is currently a topic of great interest concerning both nanomedical applications and nano-safty issues. However, a deep knowledge about the molecular mechanisms that drive these interactions is still lacking. To contribute improving the understanding of these interactions, several techniques (dynamic light scattering, microscopy, nuclear magnetic resonance, fluorescence and UV-Vis spectroscopy and quartz crystal microbalance) were exploited to elucidate the characteristics of protein-NP systems. β2m was chosen as a convenient model for amyloidogenic proteins that may be quite unstable polypeptides with a high tendency to misfold and aggregate into fibrils. Among the nanoparticles, AuNPs were selected for their versatility and their widespread application. AuNPs with different organic shell composition were synthesized, characterized and tested mainly with β2m wild-type and in some cases also with two more amyloidogenic variants, i.e. ΔN6 and D76N. The obtained results showed that the nature of the organic shell and the dimensions of AuNPs play a critical role in determining the nature of the interaction. It has been found that 7.5 nm citrate-stabilized AuNPs (Cit-AuNPs) form transient adducts with β2m that proved capable of reducing protein association and aggregation and, as far as ΔN6 variant is concerned, to avoid intrinsic protein partial unfolding. Furthermore, Cit-AuNPs were able to partially hamper D76N fibrillogenesis in vitro. AuNPs coated with 6-mercaptohexanoic acid (MHA), (11-mercaptoundecyl)-N,N,N-trimethylammonium (MUTAB) were prepared starting from Cit-AuNPs and smaller 3-mercaptopropionic acid (MPA) AuNPs were obtained through a direct synthesis. It was found that MHA-AuNPs caused β2m unfolding and its precipitation in protein-NP large agglomerates. On the other hand, MUTAB-AuNPs in presence of β2m irreversibly aggregate while the overall protein structure was preserved except for some minimal conformational changes. With the smaller MPA-AuNPs β2m formed dynamic complexes through a highly localized patch maintaining mainly its native structure. These results underline once again the complexity and the versatility of protein-NP systems, but also suggest the possibility to exploit these interactions to interfere with physiological or pathological processes. Regardless the specific results obtained with the particular protein-NP system considered, in this thesis the inadequacy of some techniques to describe quantitatively protein-NP interactions and the care that should be taken in the interpretation of the results are highlighted.